Electronic Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2013 Supporting Information The force of transporting amino-acid into the living cell measured by atomic force microscopy Xin Shanga,‡, Yuping Shanb,‡, Yangang Pan a,‡, Mingjun Caia, Junguang Jianga, Hongda Wang a,* aState Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, P.R. China, bDepartment of Physics, Florida International University, Miami, FL 33199, USA * Corresponding author: H. Wang, E-mail: [email protected] ‡ X Shang, Y Shan and Y Pan contributed equally to this paper Electronic Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2013 Illustration of transporting cysteine into the cell Figure S1. (a) Scheme of transporting the cysteine on AFM tip into the cell via the amino acid transporter. (b) The surface chemistry functionalization of AFM tip with cysteine. PEG linker was used to attach the cysteine on the AFM tip. Electronic Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2013 Morphology of HeLa cells in HBSS buffer solution HeLa cells, the well-known cancer cells that proliferated rapidly，were used to perform AFM force measurements. The living HeLa cells in Hanks’s BSS buffer were imaged by AFM. Figure S2a and Figure S2b show the topography and amplitude images of living HeLa cells growing on glass slides for 24 h. The height of cells measured by AFM is about 2 μm, which is consistent with the height of the cellular monolayer reported in references from AFM imaging1. The actual height of cells obtained from fluorescence microscopy is about 8-10 μm. Clearly, the AFM tip deformed the cells during imaging but not destroyed them. The region around the nuclei appeared to be the cell membranes and cytoplasm, which was chosen for AFM force measurements. Figure S2. Morphology of living HeLa cells. (a) Topographical image of living cells. The bright regions of the image are the nuclei. (b) The corresponding amplitude image of living cells. The scale bar is 10 μm. Electronic Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2013 In-situ trypan blue dye test of HeLa cells To verify the survivability and plasma membrane integrity of HeLa cells during the AFM force measurements, we did the in-situ trypan blue dye test. The AFM force experiment was performed on adherent HeLa cells in the definite regions. After up to 50-100 force-distance cycles were recorded for each cell, the trypan blue dye test were in situ done on the same cells. Figure S3a and Figure S3b show the optical image of HeLa cells before AFM force experiment and after AFM force experiment staining with trypan blue. After the AFM force experiments, there was no significantly difference in morphology and survivability compared with the control experiment (Figure S3c, Figure S3d and Figure S3f). The results indicate that the cells lived in good conditions during the AFM experiments. Electronic Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2013 Figure S3. Trypan blue dye test of HeLa cells. (a) Optical images of HeLa cells before force measurement. The black lines on the top and right of the image were the markers to locate the position of cells. (b) Optical images of HeLa cells after force measurement and staining with trypan blue. (c) Optical images of control HeLa cells before AFM experiment. The black lines on the top and left of the image were the markers. (d) Optical images of control HeLa cells staining with trypan blue. (e) Optical images of positioning the AFM cantilever on the cell monolayers with the assistance of a CCD camera. (f) The percentage of survivability from the control and force measurement. 700 cells were counted for both force experiment and control experiment. The scale bar is 50 μm. Electronic Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2013 Control experiments for the specificity of transporting events To test the specificity of blocking experiments, we tried to block the transporting events by non-related amino acids. We performed force measurements with cysteine-coated tip via NHS- PEG-MAL cross-linker on HeLa cells in HBSS buffer, and then 0.1 mM phenylalanine or tryptophane were injected into the solution. The results reveal that binding probability don’t change significantly by the addition of free non-related amino acid (Figure S4a and Figure S4b). To determine whether the linker’s end groups (MAL or Aldehyde) contribute to the binding events observed in Figure 2e and Figure 2f, we performed control experiments by linker- modified AFM tips without amino acids. There was no force signal either with NHS-PEG-MAL- modified tip or NHS-PEG-aldehyde-modified tip (Figure S4c and Figure S4d). The results indicate that the peaks observed in Figure 2e and Figure 2f were not from the contribution of the linkers. Electronic Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2013 Figure S4. Control experiments to verify the specificity of transporting event. Binding probabilities of cysteine-coated AFM tips on HeLa cells without (left) and with free phenylalanine ((a), right bar) or tryptophane ((b), right bar) in solution. Values are mean ± s.e.m., n=1000-2000. (c) The force curve with a NHS-PEG-MAL-modified tip on living HeLa cells in the HBSS buffer. (d) The force curve with a NHS-PEG-aldehyde-modified tip on living HeLa cells in the HBSS buffer. Electronic Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2013 The hypothetical process of transporting cysteines into living cell The hypothetical process of transporting cysteines into living cells via the amino acid transporter is shown in Figure S5. Firstly, the AFM tip with a cysteine coupled to cross-linker NHS-PEG-MAL via a thiol group moves towards the outer surface of cell membranes (Figure S5a). Amino acid transporter specifically recognizes and binds the cysteine (Figure S5b), and subsequently the transporter changes their conformations to transport the amino acid from the extracellular medium into the cell (Figure S5c). With the tip retraction from the cell, the cysteine is pulled out of cell membranes (Figure S5d). In contrast, while the AFM tip carrying a cysteine coupled to the cross-linker aldehyde-PEG-NHS via α-amino group moves towards the outer surface of cell membranes (Figure S5a’), the cysteine binds to the amino acid transporter without the following translocation process happening (Figure S5b’). Finally, the AFM tip is drawn away from the cell (Figure S5c’). Our results support the “two-step” hypothesis about amino acid translocation2, and suggest that the conformation of cysteine in the presence of free α-amino group is important for discriminating the substrate for translocation. The scheme (Figure S5) just shows a simple concept about the process of transporting amino acid on the tip into the cell, which can be easily understood about the concept of “fishing” emphasized in this work. For the real process that could be more complicated, the amino acid is very small compared to the AFM tip, so that the amino acids may be also attached to the AFM tip sidewall. The cell membranes are very soft, the AFM tip sidewall is not absolutely smooth, and there are also water molecules between the cell membranes and the tip, therefore there should be many contacting points between the tip sidewall and cell membranes for amino acid transporting. While the apex of AFM tip touched the cell membranes, the amino acid on the side wall of AFM tip could interact with the soft cell membranes and be transported. In our experiments, we could also observe the Electronic Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2013 force curves that the unbinding force “fu” appears during the contact between the AFM tip and cell membranes. This result indicates that the amino acid on the AFM tip could be transported after the apex of AFM tip contacting with the cell membranes, and the amino acid may be detached from the cell membranes before the end of AFM tip departing from the cell, which totally supports our thought that the amino acid may be on the sidewall of AFM tip. Figure S5. Scheme of the interactions between the cysteine functionalized onto AFM tip via two types of linkers and the amino acid transporter in the living cell membranes. The blue parts represent the amino acid transporter. The yellow tetragon represents the thiol group of cysteine. The purple ball represents the α-amino group of cysteine. The green triangle represents the α- carboxyl group of cysteine. (a) The AFM tip modified with cysteine which is coupled to cross- linker NHS-PEG-MAL via a thiol group, moves towards the cell membrane surface. (b) The cysteine binds to amino acid transporter. (c) The cysteine is transported into the cell. (d) The cysteine is pulled out of the cell. (a’) The AFM tip modified with cysteine which is coupled to cross-linker aldehyde-PEG-NHS via α-amino group, moves towards the cell membrane surface. (b’) The cysteine binds to amino acid transporter. (c’) The cysteine is drawn away from the cell. Electronic Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2013 The criterion to select the force events We chose S/N (signal/noise) ＞3 as the criterion to select the force peaks. For example, we considered the force events in Figure S6a and S6c as the obvious peaks; however, the force events in Figure S6b and S6d were considered as no peak.